TRYPTOPHAN-FREE ESCHERICHIA-COLI F1-ATPASE

We have engineered a mutant form of Escherichia coli F-1-ATPase which is tryptophan-free and contains five mutations, namely delta W28L/alph a W513F/gamma W108Y/gamma W206Y/ beta W107F. A strain carrying all fiv e mutations grew normally by oxidative phosphorylation. Purified mutan t F-1-ATPase showed V-max and K-m both 65% higher than wildtype, resul ting in K-cat/K-m the same as wild-type. The pH dependence of ATPase a ctivity in mutant enzyme was very similar to that in wild-type. Cataly tic-site nucleotide-binding characteristics were measured using the an alog lin-benzo-ADP and sensitivity to inhibitors was tested using dicy clohexylcarbodiimide, azide and aurovertin. The mutant enzyme was very similar to wild-type in each of these characteristics. The fluorescen ce spectrum of mutant enzyme confirmed the absence of tryptophan. We h ave therefore established that it is possible to generate a tryptophan -free enzyme which retains normal catalytic function, oligomeric stabi lity and in vivo assembly. (C) 1994 Academic Press, Inc.